circCDK13-loaded small extracellular vesicles accelerate healing in preclinical diabetic wound models

Chronic wounds are a major complication in patients with diabetes. Here, we identify a therapeutic circRNA and load it into small extracellular vesicles (sEVs) to treat diabetic wounds in preclinical models. We show that circCDK13 can stimulate the proliferation and migration of human dermal fibroblasts and human epidermal keratinocytes by interacting with insulin-like growth factor 2 mRNA binding protein 3 in an N6-Methyladenosine-dependent manner to enhance CD44 and c-MYC expression. We engineered sEVs that overexpress circCDK13 and show that local subcutaneous injection into male db/db diabetic mouse wounds and wounds of streptozotocin-induced type I male diabetic rats could accelerate wound healing and skin appendage regeneration. Our study demonstrates that the delivery of circCDK13 in sEVs may present an option for diabetic wound treatment.

4. The statement in "line 170-175" about the translafion is not accurate.The authors drew the conclusion that "the observed promofion of proliferafion and migrafion mediated by circCDK13 is not aftributed to the encoded protein CDK13" based on the results that CDK13 protein (at 164 kD) levels were not changed by overexpression or knockdown of circCDK13.However, the authors should know that the MW of circRNA encoded proteins should be smaller than the full-length mRNA encoded proteins.If the author would like to claim that the cell funcfion changes were not mediated by circCDK13 encoded protein, the author should not examine the CDK13 protein at 164kD but should predict the possible MW of circCDK13 encoded protein and examine the proteins at the predicted MW using CDK13 anfibodies.
5. The concentrafion and durafion of cycloheximide treatment were not clear stated in the Methods or Figure legends secfion.6.It is stated in line 271 that "circCDK13 and IGF2BP3 synergisfically enhanced the stability of CD44 and c-MYC mRNA".I wonder whether the authors examined whether circCDK13 or IGF2BP3 directly bind with CD44 and c-MYC mRNA.If not, how circCDK13 and IGF2BP3 affected the stability of CD44 and c-MYC mRNA?Is there any mediator involved in?
Reviewer #2 (Remarks to the Author): This study aims to idenfify a novel circular RNA (circRNA) loaded into small extracellular vesicles (sEVs) to reverse the impaired wound healing caused by diabetes.
Ufilizing an external database, the invesfigators idenfified circCDK13 that was downregulated in human diabefic wounds.In support of circCDK13, they show deplefion of circCDK13 reduces the migrafion and proliferafion of human dermal fibroblasts (HDF) and human epidermal kerafinocytes, and overexpression showed the opposite.At a mechanisfic level, circCDK13 interacted directly with IGF2BP3 in an n6methyladenosine manner to increase the expression of CD44 and c-Myc.Encapsulafion of mesenchymal stem cell-derived small extracellular vesicles (sEVs) that overexpress circCDK13 (circCDK13OE-sEVs) reversed the effects of experimental hyperglycemia (Age-BSA) on the migrafion and proliferafion of HDFs and HEKs.circCDK13OE-sEVs were more effecfive than N-sEVs in accelerafing wound healing and skin appendage regenerafion in db/db type 2 diabefic rats and Streptozotocin (STZ)-induced type I diabefic rats.
The experiments showing how circCDK13 interacts with IGF2BP3 and their effects on HDFs and HEKs under hyperglycemic condifions were elegant and convincing.While the circCDK13OE-sEVs appeared to increase the wound healing rate, the design and interpretafion raised concerns.Given that the primary goal of these experiments was the development of a therapeufic to reverse wound healing in diabefics, these experiments need to be more detailed.
1.It is stated that circCDK13 was downregulated in "diabefic wounds compared with acute wounds".Do you mean non-diabefic wounds, or are you referring to acute or chronic diabefic wounds? 2. The opfimal experimental wound is stented because rats have a subcutaneous muscle, the panniculus carnosum, that causes the wound to heal by contracfion.Stenfing the wound promotes a healing paftern more typical of that seen in humans 3. None of the wounding experiments were performed to complete healing, which is the proper standard.This is important for several reasons.The primary goal of these experiments is to evaluate a therapeufic intervenfion.The accelerafion of the wound healing rate measured in days is the benchmark to determine if an intervenfion jusfifies its cost.None of the experiments contained a wild-type rat control group.
4. Wound healing progresses between mulfiple defined stages: proliferafive, angiogenic and remodeling.Understanding the role of circCDK13 during these stages would greatly enhance the significance of the experiments.
5. The wound healing rate in the streptozocin-induced type 1 diabetes was only carried out for 14 days.Why?According to the protocol, the intervenfion was administered unfil day 17.Why exclude that dose?Moreover, the differences in healing rates were very similar in the N-sEVs and the circCDK13OE-sEVs suggesfing a minimal therapeufic effect.

6.
Given the in vivo model, why not measure the expression of circCDK13 during the different stages of wound healing in diabefic and wild-type rats?Or use a siRNA against circCDK13 in wild-type rats?
Reviewer #3 (Remarks to the Author): In the present study, author revealed that impaired healing of diabefic wounds was fightly associated with the downregulafion of circCDK13, and then confirmed a novel wound healing-promofing mechanism that circCDK13 directly interacted with IGF2BP3 to form a circRNA-protein-mRNA ternary complex, which synergisfically enhanced the stability of CD44 and c-MYC mRNA to promote the proliferafion and migrafion of HDFs and HEKs.Author successfully constructed engineered sEVs bearing circCDK13 and corroborated that circCDK13OE-sEVs.Author confirmed that in the wounds of db/db diabefic mice and STZ-induced type Ⅰ diabefic rats, circCDK13OE-sEVs could accelerate reepithelializafion and granulafion fissue formafion, and promote wound remodeling and regenerafion of skin appendages.
2. The authors perform immunoprecipitafion of circCDK13 followed by mass-spectrometry to idenfify protein interactors.These data are not shown: Fig. S7 just shows the LC-MS/MS plot, while a supplementary table with the idenfified interactors should be provided.(e.g., kidneys, etc.)   4. The authors should examine the effect of overexpression or knockdown of circCDK13 on cell death (such as autophagy, apoptosis, necrosis, etc.) in HDFs and HEKs.

Authors should examine studies of circCDK13OE-sEVs on funcfion in other organs
5. Other studies on circCDK13 have shown that acfivafion of endogenous CDK13 can promote the expression of circCDK13, and the high expression of circCDK13 promotes the occurrence and development of prostate cancer, while inhibifing the expression of circCDK13 can reduce the occurrence of prostate cancer.Authors should consider whether the expression of circCDK13 might produce tumorigenicity.。6.The authors should increase the detecfion of whether circCDK13 affects classical proliferafion pathways (such as Yap).
7. Macrophages play an indispensable role in the process of wound repair in the body.The authors should examine the expression of circCDK13 and IGF2BP3 in macrophages and its influence on them.
8. Most chronic wounds are characterized by a large number of inflammatory cell infiltrafion, leading to overexpression of reacfive oxygen species and MMP.It is suggested that the authors examine the effects of overexpression or knockdown of circCDK13 on the expression of reacfive oxygen species and MMP. 9. Please revise the English grammar and wrifing style of the manuscript，spelling and grammafical errors should be excluded.

Point-by-point responses to the comments from Reviewers
We sincerely thank all the Reviewers for their constructive comments and helpful suggestions.We have done our best to address all the issues raised and hope that our revised manuscript now meets your expectations

Reviewer #1:
In the present study, Huang et al. proposed a novel treatment strategy for diabetic wound by investigating the possible role of certain circular RNAs in the pathogenesis and treatment of wound healing in diabetic conditions.The authors presented data showing that circCDK13 was downregulated in diabetic wounds.Depletion of circCDK13 in human dermal fibroblasts and human epidermal keratinocytes inhibited the capacities of their proliferation and migration.In animal models, engineered small extracellular vesicles with overexpression of circCDK13 could accelerate the wound healing process in diabetic animals.The idea was relative novel and the research was with good experimental design.However, the study heavily replied on overexpression of the circular RNA embedded in the extracellular vesicles.It is not clear for the physiological function of this circular RNA in wound repair.Here are some additional comments.
Response: Thanks very much for your high evaluation.
1.For rationale, the authors analyzed the microarray data and found 111 circRNAs including circCDK13 were downregulated.However, it was not explained why circCDK13 was chosen for further investigation.Is circCDK13 the most decreased circRNA among these 111?Response: Thanks very much for reviewer's question.The reasons for choosing circCDK13 for in-depth study in this study are as follows.Firstly, we analyzed a set of microarray data (GSE114248) published by Wang[1], and found that 111 circRNAs including circCDK13 were down-regulated in the diabetic foot ulcer (DFU) compared with normal wounds (NWs).Secondly, we carried out homology analysis on the top 40 circRNAs with the highest expression differences and found that 6 circRNAs shared homology between Homo sapiens and mice.Thirdly, we used AGE-BSA, an alternative to AGEs commonly used to simulate the diabetic wound microenvironment, to intervene HEKs and HDFs and found that three circRNAs, including circCDK13, were significantly downregulated in the AGE-BSA intervention group.Subsequently, we used siRNA interference technology to knock down the intracellular expression abundance of above three circRNAs and observed that the proliferation and migration of HEKs and HDFs were significantly reduced only by knocking down circCDK13.In addition，circCDK13 was also reported to play a significant role in the proliferation, invasion, and metastasis of cancer cells [2].So, in our study, circCDK13 was chosen for further investigation.The results were described on Page 5 Line 114-Page 6 Line 139 and screening process of circCDK13 was shown in Supplementary Fig. 1c.

CircCDK13 was chosen as a promising therapeutic agent for diabetic wounds
CircRNAs have been discovered as powerful actors of gene expression with critical functions in many diseases.To elucidate the role of circRNAs in the healing process of diabetic wounds, we analyzed a set of microarray data (GSE114248) published by Wang 8 , and found that 111 circRNAs including circCDK13 were down-regulated in the DFU compared with normal wounds (NWs) (Supplementary Fig. 1a).Heat map of the top 40 circRNAs was shown in Fig. 1a and normalized expression level of circCDK13 were calculated with the microarray data (Fig. 1b).Homology analysis was performed on the top 40 circRNAs and we found that 6 circRNAs including circCDK13 shared homology between Homo sapiens and mice.The homologous sequence of circCDK13 was shown in Supplementary Fig. 1b.Additionally, the lower expression of circCDK13 was also observed in the wounds of diabetic mice (Fig. 1c).To simulate the microenvironment of diabetic wounds in vitro, AGE-BSA, an alternative to AGEs commonly used in research, was produced by co-incubating D-glucose with bovine serum albumin (BSA) at 37°C for 2 months (Supplementary Fig. 2a-c).After treated with AGE-BSA at different concentrations, HDFs and HEKs exhibited the decreased proliferation assessed by CCK8 assay (Fig. 1d, e) and migration assessed by scratching test (HDFs: Supplementary Fig. 3a, Fig. 1f; HEKs: Supplementary Fig. 4a, Fig. 1g) and transwell assay (HDFs: Supplementary Fig. 3b, Fig. 1h; HEKs: Supplementary Fig. 4b, Fig. 1i).Among above 6 circRNAs, we found that three circRNAs, including circCDK13, were significantly downregulated in AGE-BSA treated HDFs and HEKs.
Fig. 1j, k demonstrated the downregulation of circCDK13 in AGE-BSA-treated HDFs and HEKs.Among the three downregulated circRNAs, circCDK13 was reported to play a significant role in the regulation of cellular proliferation and migration 18 .Therefore, we speculated that the downregulation of circCDK13 might be involved in the mechanisms of impaired wound healing in diabetes, implying circCDK13 was a promising therapeutic agent for diabetic wounds.The screening process of circCDK13 was summarized in Supplementary Fig. AGE-BSA group and control group (AGE-BSA concentration of 0 μg/ml).To avoid misunderstanding, AGE-BSA concentration of 0 μg/ml group has been changed to control group in Fig. 1j, k.Sanger sequencing following PCR was used to show the "head-to-tail" splicing of circCDK13 and the Sanger sequencing of back splice junction site of circCDK13 can be found in Fig. 1l, lower right panel.To match the description in the results and Fig. 1n-q, we changed CDK13 to CDK13 mRNA in Fig. 1n-q.
Additionally, to better understand the description of the results, we reordered Fig. 1p-t as following and rewrote the description of Fig. 1a-k  Actinomycin D assay (Fig. 1n, o) and RNase R exonuclease digestion assay (Fig. 1p, q) further indicated that circCDK13 was more stable than CDK13 mRNA.Furthermore, fluorescence in situ hybridization (FISH) (Fig. 1r) and nuclear and cytoplasmic fractionation analysis (Fig. 1s, t) demonstrated that circCDK13 was predominantly localized in the cytoplasm.Collectively, these results implicated that circCDK13 in HDFs and HEKs is a stable circRNA generated from CDK13 by back-splicing.
CCK-8 assay was performed to show that the knockdown of circCDK13 resulted in the decreased viability of HDFs (Fig. 2d) and HEKs (Fig. 2e).Next, an overexpression vector of circCDK13 was constructed (Supplementary Fig. 5a, b) and then transfected into HDFs and HEKs (Fig. 2f) to overexpress circCDK13 (Fig. 2g, h).The results of CCK-8 assay show that the enforced expression of circCDK13 dramatically enhanced the viability of HDFs (Fig. 2i) and HEKs (Fig. 2j).Furthermore, the percentage of EdUpositive cells was decreased by circCDK13 knockdown and increased by circCDK13 overexpression in HDFs (Fig. 2k, Supplementary Fig. 5c) and HEKs (Fig. 2l, Supplementary Fig. 5d).Subsequently, scratch and transwell assays were performed to investigate the effect of circCDK13 on cell migration.The migration area of circCDK13 knockdown group was remarkably lower than that of its control group and the migration area of circCDK13 overexpression group was higher than that of corresponding control group (Fig. 2m, n; Supplementary Fig. 5e, f).Additionally, the results of transwell assay demonstrated that circCDK13 knockdown decreased and circCDK13 overexpression increased the number of migrated cells (Fig. 2o, p; Supplementary Fig. 5g, h).Response: Thank you for your careful review.We strongly agree with your opinion.
The conclusion that "the observed promotion of proliferation and migration mediated by circCDK13 is not attributed to the encoded protein CDK13" based on the results that CDK13 protein (at 164 kD) levels were not changed by overexpression or knockdown of circCDK13 is not accurate.As you said, if circCDK13 can encode protein CDK13, the MW of circRNA encoded proteins should be smaller than the full-length mRNA encoded proteins.However, in literature, to date, there have been no reports of circCDK13-encoded protein, which needs an in-depth study.In our study, we detected the CDK13 mRNA and protein expressions (Supplementary Fig. 6) and found that they were unaffected by siRNAs and overexpression vector of circCDK13.The results demonstrated that siRNAs used in our study didn't degrade CDK13 linear mRNA Moreover, we detected the CDK13 mRNA (Supplementary Fig. 6a-d) and protein expressions (Supplementary Fig. 6e) and found that they were unaffected by siRNAs and overexpression vector of circCDK13.The results demonstrated that siRNAs used in our study didn't degrade CDK13 linear mRNA transcript and circCDK13 couldn't increase expression of CDK13 mRNA and protein by a positive feedback pathway reported before 18 .Collectively, the above findings indicated that circCDK13 rather than CDK13 protein played an essential role in regulating the proliferation and migration of HDFs and HEKs.To further prove that circCDK13 and IGF2BP3 synergistically promote the expression of CD44 and c-MYC by enhancing the stability of CD44 and c-MYC mRNA, we performed the PCR analysis of pull-down products precipitated by circCDK13 probe or IGF2BP3 antibody.As expected, we found that there was an enrichment of CD44 and c-MYC mRNA in the pull-down products (Supplementary Fig. 14), which provided the possibility for circCDK13 and IGF2BP3 to bind with CD44 and c-MYC mRNA for stabilization.However, it's unclear whether circCDK13 or IGF2BP3 directly binds to CD44 and c-MYC mRNA and whether other mediators are involved in the binding of circCDK13-IGF2BP3 complex to CD44 and c-MYC mRNA.One study reported that IGF2BP3 could increase c-MYC mRNA stability by binding to the coding region instability determinant (CRD) residing in the 3′-terminus of the c-MYC coding region 26 .

References
In another study, IGF2BP3 was found to bind to the 3'-UTR of CD44 mRNA for its stabilization 29 .Combining literature reports and our results, we speculated that circCDK13 and IGF2BP3 synergistically promoted the expression of CD44 and c-MYC by enhancing the stability of CD44 and c-MYC mRNA in HDFs and HEKs.Furthermore, we observed significantly lower expression levels of IGF2BP3, CD44, and c-MYC proteins in epidermal keratiocytes and dermal fibroblasts from the DFU group compared with the NWs group (Fig. 4i).Based on the above experimental results, we inferred that circCDK13 and IGF2BP3 synergistically enhance the stability of CD44 and c-MYC mRNA, increase CD44 and c-MYC protein levels, and thereby enhance the proliferation and migration of HDFs and HEKs.

Reviewer #2:
This study aims to identify a novel circular RNA (circRNA) loaded into small extracellular vesicles (sEVs) to reverse the impaired wound healing caused by diabetes.
Utilizing an external database, the investigators identified circCDK13 that was downregulated in human diabetic wounds.In support of circCDK13, they show depletion of circCDK13 reduces the migration and proliferation of human dermal fibroblasts (HDF) and human epidermal keratinocytes, and overexpression showed the opposite.At a mechanistic level, circCDK13 interacted directly with IGF2BP3 in an n6-methyladenosine manner to increase the expression of CD44 and c-Myc.
Encapsulation of mesenchymal stem cell-derived small extracellular vesicles (sEVs) that overexpress circCDK13 (circCDK13OE-sEVs) reversed the effects of experimental hyperglycemia (Age-BSA) on the migration and proliferation of HDFs and HEKs.circCDK13OE-sEVs were more effective than N-sEVs in accelerating wound healing and skin appendage regeneration in db/db type 2 diabetic rats and Streptozotocin (STZ)-induced type I diabetic rats.

The experiments showing how circCDK13 interacts with IGF2BP3 and their effects on
HDFs and HEKs under hyperglycemic conditions were elegant and convincing.While the circCDK13 OE -sEVs appeared to increase the wound healing rate, the design and interpretation raised concerns.Given that the primary goal of these experiments was the development of a therapeutic to reverse wound healing in diabetics, these experiments need to be more detailed.
Response: Thanks very much for your high evaluation.

It is stated that circCDK13 was downregulated in "diabetic wounds compared with acute wounds". Do you mean non-diabetic wounds, or are you referring to acute or chronic diabetic wounds?
Response: Thank you for your question.In this study, "acute wounds" refer to nondiabetic wounds.To avoid confusion, we have replaced "acute wounds" with "nondiabetic wounds" in the abstract on Page 2 Line 36.
2. The optimal experimental wound is stented because rats have a subcutaneous muscle, the panniculus carnosum, that causes the wound to heal by contraction.
Stenting the wound promotes a healing pattern more typical of that seen in humans.
Response: Thank you for your suggestion.Yes, it's very reasonable to stent the experimental wounds for promoting a healing pattern more typical of that seen in humans.In our study, rubber rings (Fig. 6b, Fig. 7b) were attached to the edge of the wounds, which effectively prevented the contraction of the wounds.showing a shorter wound length in circCDK13 OE -sEVs group (e, f).Furthermore, in tissue sections, more skin appendages, such as hair follicles and sebaceous glands, were observed in circCDK13 OE -sEVs-treated wounds on day 14 (e).By comparing the data from diabetic rats and wild-type rats, we found that the wound areas of diabetic rats were larger than that of wild-type rats at the same time and with the same treatment.Response: Thank you for your valuable suggestion.As you said, the wound healing process can be divided into multiple defined stages including inflammatory phase, proliferative phase, and remodeling phase.In our study, we observed the positive role of circCDK13 in proliferative phase.Especially, we observed that circCDK13 promoted not only the diabetic wound healing but also the regeneration of skin appendages such as hair follicles and sebaceous glands, which enhanced the significance of circCDK13 application in wound treatment.As for the role of circCDK13 in inflammatory phase and remodeling phase needs to be further investigated.
5. The wound healing rate in the streptozocin-induced type 1 diabetes was only carried out for 14 days.Why?According to the protocol, the intervention was administered until day 17.Why exclude that dose?Moreover, the differences in healing rates were very similar in the N-sEVs and the circCDK13 OE -sEVs suggesting a minimal therapeutic effect.
Response: Thank you for your suggestion.We are sorry for causing confusion.As you required, the rate of wound healing on the 17th day after the operation has been added in Fig. 7g.The data exhibition form of Fig. 7g may make you feel that the healing rates are similar in N-sEVs and circCDK13 OE -sEVs groups, but in fact there are significant differences in healing rates between two groups as shown as histogram on the 14th day (for example) by statistics.Response: Thank you for your insightful review and valuable suggestion.As you required, we used the siRNAs against circCDK13 in wild-type rats and measure the expression of circCDK13 during the different stages of wound healing in diabetic and wild-type rats.Two full-thickness skin wounds were created on the back of each wildtype rat and then the siRNAs were used to knock down the expression of circCDK13 in the wound tissue.The results showed that circCDK13 knockdown decreased the wound healing rate, as determined by lager wound areas measured on day 3, 7, and 14 post-wounding compared with control groups (a-e).H&E staining was conducted to evaluate the wound length in each experimental group and the results were consistent with the above wound area measurements (f, g).Furthermore, fewer skin appendages, such as hair follicles and sebaceous glands, were observed in circCDK13 knockdown wounds on day 14 (h).Additionally, the expression of circCDK13 was measured during the different stages of wound healing in diabetic and wild-type rats.At the designed time points, we found that the expression of circCDK13 in the wound tissue of diabetic rats was significantly reduced compared with the wounds of wild-type rats (i).In summary, the decrease of circCDK13 in skin wounds resulted in slow wound healing.

Reviewer #3:
In the present study, author revealed that impaired healing of diabetic wounds was tightly associated with the downregulation of circCDK13, and then confirmed a novel wound healing-promoting mechanism that circCDK13 directly interacted with IGF2BP3 to form a circRNA-protein-mRNA ternary complex, which synergistically enhanced the stability of CD44 and c-MYC mRNA to promote the proliferation and migration of HDFs and HEKs.Author successfully constructed engineered sEVs bearing circCDK13 and corroborated that circCDK13 OE -sEVs.Author confirmed that in the wounds of db/db diabetic mice and STZ-induced type Ⅰ diabetic rats, circCDK13 OE -sEVs could accelerate re-epithelialization and granulation tissue formation, and promote wound remodeling and regeneration of skin appendages.
Response: Thanks very much for your high evaluation.
1.The co-localization experiments of circCDK13 and IGF2BP3 using Fish showed that circCDK13 and IGF2BP3 interact.Please provide more data to support this finding.
Response: Thank you for your suggestion.As you required, the binding between circCDK13 and IGF2BP3 was confirmed by RNA pull-down and RIP-qPCR assays (Fig. 3h-j).Moreover, we carried out an RNA FISH-immunofluorescence (FISH-IF) analysis and found that circCDK13 colocalized with IGF2BP3 in the cytoplasm of HDFs and HEKs (Fig. 3k).All above results showed that circCDK13 and IGF2BP3 interacted with each other.
2. The authors perform immunoprecipitation of circCDK13 followed by massspectrometry to identify protein interactors.These data are not shown: Fig. S7 just shows the LC-MS/MS plot, while a supplementary table with the identified interactors should be provided.
Response: Thank you for your valuable suggestion.As you required, a supplementary table containing the identified interactors has been supplemented in the Supplementary Material.In this study, we found that circCDK13 expression was significantly downregulated in diabetic wound tissue compared to normal wound tissue, and confirmed that circCDK13 depletion and overexpression in HDFs and HEKs decrease and increase, respectively, cell proliferation and migration.Therefore, we hypothesized that the delivery of circCDK13 by sEVs would be able to compensate for the lack of circCDK13 in HDF and HEK in diabetic wounds, thereby promoting diabetic wound healing.Changes in the expression level of circCDK13 in cells were analyzed by RT-qPCR when circCDK13 OE -sEVs was co-incubated with HDFs or HEKs.We found that intracellular circCDK13 levels increased significantly after 6 h and 12 h of exposure to circCDK13 OE -sEVs, and then the abundance of circCDK13 gradually decreased over time (a, b).The abundance of circCDK13 was close to that in control cells after 120 h (a, b).Exogenous circRNAs can be degraded in cells, although circRNAs is more stable than linear RNAs.

Authors should examine studies of circCDK13
In addition, we found that knockdown or overexpression of circCDK13 in HDFs and HEKs did not alter the expression levels of CDK13 protein (Supplementary Fig. Statistical significance is indicated as follows: NS, not significant.9. Please revise the English grammar and writing style of the manuscript，spelling and grammatical errors should be excluded.
Response: Thanks for your suggestion.We are very sorry for the spelling and grammatical errors.To revise the English grammar and writing style of the manuscript, we asked our colleague whose native language is English to review our revised manuscript.We hope our new manuscript will meet the requirement for publication.
figure file.
Fig. 2 was reordered and Figure Legend was rewritten.Some results were separated

Fig. 2
Fig.2 and Fig.2 legend: transcript and circCDK13 couldn't increase expression of CDK13 mRNA and protein by a positive feedback pathway reported before[1].Collectively, the above findings indicated that circCDK13 rather than CDK13 protein played an essential role in regulating the proliferation and migration of HDFs and HEKs.The results were described on Page 8 Line 174-Page 8 Line 181.Page 8 Line 174-Page 8 Line 181:

[ 1 ]
Fig. 3t: The protein level of IGF2BP3 in HDFs and HEKs with knockdown or

Fig
Fig. 7g:Histogram on the 14th day: resulted in slow wound healing.(a) Representative images of the wound area by different treatments on days 0, 3, 7, and 14, after operation.Scale bar, 1 mm.(b) Simulation plots of the wound closure areas.Scale bar, 1 mm.(c-e) Quantitative evaluation of the wound closure rate (n=6).(f) Representative images of H&E staining of wound sections on day 7. Scale bar, 1 mm.(g) Quantitative analysis of wound width (n=6).(h) Representative images of H&E staining of wound sections on day 14.Scale bar, 200 μm.(i) RT-qPCR analysis of circCDK13 expression abundance in normal skin of wild-type rats (Normal skin), wounds of wild-type rats (Normal wound), and wound of diabetic rats (Diabetic wound) at different time points (N=6).Data are represented as mean ± SD.Statistical significance is indicated as follows: NS, not significant，*p < 0.05, **p < 0.01, ***p < 0.001.Compare to Norma skin group，### P<0.001.
OE -sEVs on function in otherorgans (e.g., kidneys, etc.) Response: Thank you for your valuable suggestion.As you required, we examined whether circCDK13 OE -sEVs could function on other organs including the lungs, liver, spleen, kidney, and heart of the rats after multiple administrations of N-sEVs or circCDK13 OE -sEVs.H&E staining of the heart, lung, liver, kidney, and spleen in circCDK13 OE -sEVs group demonstrated no histological abnormalities or immune cell infiltration in comparison with those treated with N-sEVs or PBS.Therefore, we initially believe that multiple administrations of N-sEVs or circCDK13 OE -sEVs to skin wounds are safe in vivo and have no negative impact on the vital organs.Effects of circCDK13 OE -sEVs on other organs in ratsRepresentative images of H&E staining of other organs in rats, such as lungs, liver, spleen, kidney and heart.scale bar, 40 μm.Thanks for your valuable suggestion.As you required, we evaluated the effects of circCDK13 on autophagy, apoptosis, and necrosis of HDFs and HEKs with knockdown or overexpression of circCDK13.The results of Monodansylcadaverine (MDC) method showed that circCDK13 did not significantly affect autophagy levels (a-e).Flow cytometric analysis showed that knockdown or overexpression of circCDK13 in HDFs and HEKs did not significantly affect cell apoptosis and necrosis (f).Effect of CircCDK13 on autophagy, apoptosis, and necrosis in HDFs and HEKs(a) Monodansylcadaverine (MDC) fluorescent probe was used to detect autophagy levels in HDFs and HEKs transfected with si-NC or si-circCDK13, and vector or circCDK13-OE.scale bar, 40 μm.(b-e) After the MDC fluorescent probe has been used to label the autophagosome in HDFs and HEKs, the fluorescence intensity at 335/512 nm is measured using a fluorescence microplate reader, and the relative fluorescence intensity is finally calculated (n=6).(f) Flow cytometry analysis of apoptosis and necrosis in HDFs and HEKs transfected with si-NC or si-circCDK13, and vector or circCDK13-OE.Data are represented as mean ± SD.Statistical significance is indicated as follows: NS, not significant.Thank you for your insightful review and valuable comments.Qi et al. found that CDK13 is significantly upregulated in human prostate cancer (PCa) tissue[1].CDK13 depletion and overexpression in PCa cells decrease and increase, respectively, cell proliferation, and the pro-proliferation effect of CDK13 is strengthened by its interaction with E2F5[1].Mechanistically, transcriptional activation of endogenous CDK13, but not the forced expression of CDK13 by its expression vector, remarkably promotes E2F5 protein expression by facilitating circCDK13 formation.Further, the upregulation of E2F5 enhances CDK13 transcription and promotes circCDK13 biogenesis, which in turn sponges miR-212-5p/449a and thus relieves their repression of the E2F5 expression, subsequently leading to the upregulation of E2F5 expression and PCa cell proliferation [1].
6e), thus not forming the CDK13-circCDK13-miR-212-5p/449a-E2F5 positive feedback loop to overstimulate cell proliferation and migration.Therefore, we are confident that the administration of exogenous circCDK13 for the treatment of diabetic wounds is safe and does not lead to tumourigenesis.circCDK13 expression over time in HDFs and HEKs co-incubated with circCDK13 OE -sEVs (a, b) After co-incubation of circCDK13 OE -sEVs with HDFs or HDFs, cells were harvested at defined time points, total RNA was extracted，and then the expression level of circCDK13 was analyzed by RT-qPCR (n=3).Data are represented as mean ± SD.Statistical significance is indicated as follows: NS, not significant；**p < 0Qi JC, Yang Z, Lin T, et al.CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis.J Exp Clin Cancer Res.2021;40(1):2.Thanks for your valuable suggestion.As you required, we performed the detection of whether circCDK13 affects YAP pathway.In HDFs and HEKs, we observed that the knockdown of endogenous circCDK13 increased YAP phosphorylation levels, while upregulation of circCDK13 reduced YAP phosphorylation levels，but did not alter YAP protein expression levels.Effects of circCDK13 overexpression or knockdown on YAP pathway (a, b) The protein levels of p-YAP and YAP were detected by western blot assay in HDFs and HEKs transfected with si-NC or si-circCDK13, and vector or circCDK13-Thanks for your valuable suggestion.As you required, we examined the expressions of circCDK13 and IGF2BP3 in macrophages and its influence on them.The initial macrophages (M0) were induced to M1 macrophages or M2 macrophages, respectively.The expressions of circCDK13 and IGF2BP3 were detected in M0, M1, and M2 macrophages.Finally, we did not observe any significant difference in the levels of expression of IGF2BP3 (a) and circCDK13 (b) in macrophages with different phenotypes.Expression levels of circCDK13 and IGF2BP3 in macrophages with different phenotypes (a) The protein levels of IGF2BP3 were detected by western blot assay in macrophages with different phenotypes.(b) RT-qPCR analysis of circCDK13 abundance in macrophages with different phenotypes (n=3).Data are represented as mean ± SD.Statistical significance is indicated as follows: NS, not significant.8.Most chronic wounds are characterized by a large number of inflammatory cell infiltration, leading to overexpression of reactive oxygen species and MMP.It is suggested that the authors examine the effects of overexpression or knockdown of circCDK13 on the expression of reactive oxygen species and MMP.Response: Thank you for your valuable suggestion.In this study, we found that the expressions of reactive oxygen species (ROS), MMP2, and MMP9 were not significantly affected by knockdown or overexpression of circCDK13 in HDFs (a-c).When circCDK13 was knocked down in HEKs, the expression levels of MMP2 and MMP9 were slightly decreased (d), while when circCDK13 was overexpressed, the expression levels of MMP2 and MMP9 were slightly increased (d), but the levels of ROS did not change significantly in HEKs (e, f).Effects of circCDK13 overexpressionor knockdown on reactive oxygen species and MMP expressions (a) The protein levels of MMP9 and MMP2 were detected by western blot assay in HDFs transfected with si-NC or si-circCDK13, and vector or circCDK13-OE.(b, c)The expression levels of reactive oxygen species were measured using a fluorescence microplate reader in HDFs transfected with si-NC or si-circCDK13, and vector or circCDK13-OE (n=6).(d) The protein levels of MMP9 and MMP2 were detected by western blot assay in HEKs transfected with si-NC or si-circCDK13, and vector or circCDK13-OE.(e, f) The expression levels of reactive oxygen species were measured using a fluorescence microplate reader in HEKs transfected with si-NC or si-circCDK13, and vector or circCDK13-OE (n=6).Data are represented as mean ± SD.